大型多管水母的绿色荧光蛋白

Green fluorescent protein of the jellyfish Aequorea macrodactyla

采用PCR扩增和southern杂交筛选相结合的方法 ,从厦门水域的大型多管水母中分离到了新的绿色荧光蛋白基因 gfpxm ,并在大肠杆菌中进行了表达 .gfpxmDNA序列编码区长为 1 0 4 2bp,包含 3个外显子和两个内含子 ,cDNA编码区全长为71 7bp .gfpxmcDNA与已知野生型Aeqgfp 1 0cDNA长度一致 ,核苷酸同源性为81 9% ,推导的氨基酸序列全长为 2 38个氨基酸 ,与AeqGFP1 0的氨基酸同源性为83 6 % .将 gfpxm克隆至pTO -T7表达载体 ,GFPxm在大肠杆菌BL2 1中的表达量达菌体总蛋白的 50 %左右 .荧光性质和强度分析结果表明 ,GFPxm蛋白的激发峰为 4 76nm ,发射峰为 4 96nm ,荧光量子产率为 1 GFPxm蛋白的荧光很稳定 ,对热、碱性。

A new green fluorescent protein gene gfpxm was isolated from jellyfish Aequorea macrodactyla in the coastal region of East China Sea by the method of combination of PCR amplification and southern hybridization analysis. The gfpxm gene contains three extrons and two introns spread over 1 042 bp of genomic DNA, the entire coding region of cDNA is 717 bp, being identical to the wild type gfp Aeqgfp10 cDNA in length. The amino acid sequence of GFPxm deduced from the nucleotide sequence is 238 aa residue, sharing homology of 83 6% with that of AeqGFP10. The entire coding sequence was cloned into the pTO T7 expression vector and expressed in E coli. The expression yield of GFPxm was amounted to 50% of the total protein. Compared with GFP of A victoria, the expressed GFPxm exhibited an excitation peak at a higher wave length of 476nm and an emission peak at a lower wave length 496 nm with a higher quantum yield of 1 0. The fluorescence of GFPxm is significant stable, showing strong resistant to heat, alkaline, denaturants and salts.